Spinal cord injury (SCI) is a devastating neurological disorder without effective treatment. Astrocytes are the predominant component of the scar. There is intensive debate in the field whether astrocyte, by responding to injury microenvironment, play inhibitory or beneficial/neuroprotective roles. In our recent astrocyte RNA-Seq data of the purified GFAP+ cell from the scars of GFAP-Cre-/+Ai9-tdTomato-/+ transgenic SCI mice, we see both inhibitory and beneficial/neuroprotective factors including CSPGs, Slit proteins, integrin genes family, fibrosis-associated genes and neurotrophic factors. Which subtypes of astrocytes are these factors from? Are there temporal and ratio changes of astrocyte subtypes from the initial beneficial reactive astrocytes (RA) to the scar-forming astrocytes (SA) that express inhibitory molecules? Single-cell RNA sequencing (scRNA-Seq) of the reactive astrocytes after SCI will directly answer these critical questions and thus move the field forward. ScRNA-Seq is a highly sensitive new technology for detecting cell types/subpopulations and their gene profiles. However, scRNA-Seq has not been applied to uninjured or injured thoracic spinal cord. Furthermore, most scRNA-Seq studies have not investigated the expression of long non-coding RNA (lncRNA), a type of regulatory RNAs with important functions in the CNS. Previously, we have systematically analyzed both the protein coding gene and lncRNA expression profiles in both rat and mouse injury models at various time points after injury. In the current study, by using scRNA-Seq, we will answer the fundamental question in the field regarding reactive astrocyte constituents. First, we will characterize and quantify the cell types/subpopulations in the uninjured spinal cord and in a thoracic contusive injury epicenter using GFAP-Cre-/+Ai9-tdTomato-/+ transgenic mice, and establish astrocyte subpopulation gene marker panel (molecular signature) of both protein-coding and lncRNAs specific for each cell subpopulation. And we will localize subpopulations by in situ hybridization based on the subpopulation markers. Secondly, we will analyze the changes of activation states of astrocyte subpopulations at injury stages (acute, sub-chronic and chronic) by examining the differences in the transcriptome profiles of these subpopulations. We plan to organize single cells along activation trajectories to infer the potential sequence and origin from which reactive astrocyte subpopulations emerged in a time course, and study genes and pathways that regulate or maintain the inhibitory or beneficial astroglial states (such as neurotoxicity and neuroprotection) at different injury stages. Importantly, we will study the functional heterogeneity by FACS isolating astroglial subpopulations, and co-culturing with neurons to assess their impact on neuronal growth and guidance. Finally, we will build a publicly available SCI scRNA-Seq database that will be valuable for the research community. These studies are critical for both understanding the molecular mechanisms of astrogliosis, and for developing novel and effective therapeutic strategies to improve the injury microenvironment and axonal regeneration.